Peer-reviewed by 2 reviewers with median rating of 11.5/20. Review process was triple-blinded.

Round 1 (11.5/20)

Technical quality6

Conceptual advance and Impact8.5

General comment

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A previous study demonstrated that autism-associated variants L236S, S438P and V176I lack function in astrocytes. In this study, the authors evaluated three previously uncharacterized autism associated NHE9 variants, P117T, D496N, and Q609K and showed that all three were similar to wild-type NHE9 in endosomal expression and function when expressed in HEK293 cells. The data are nicely illustrated and the manuscript is clearly written. However, I have reservation on the experimental design.
Major: A control for the loss-of-function is missing in the functional analysis. The author should add at least any one of three previously characterized variants in this study and test whether loss-of-function can be detected in their system. The result may also argue for the concern of the cell type specificity. At this stage, I am not convinced that the results do support the conclusions.

Technical quality5

Conceptual advance and Impact3

General comment

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see comments in all the above sections.

Metadata

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Fits to the biological discipline rather the medical one.

Title

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Spell fully abbreviations in the title.
As the gene variants were not analyzed in this paper in regard to association to autism or epilepsy, authors should rephrase this in the title. As it is misleading,

Figure

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In (C) it seems as if the expression (using densitometry analysis) of the bands for D496N and Q609 variants are lower in expression comparing to the others, when normalized to tubulin. A densitometry analysis of the bands and statistical analysis would be helpful.

Introduction

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Authors should refer to polygenetics of ASD, and the very low effect sizes (or OR) of these variants. Moreover, a reference and information to the extent of the associations with ASD (and epilepsy- as indicated in the title) of the studied variants should be given.
Genes should be given in cursive (e.g. for SLC9A9).

The first sentence is not clear and seems not to be at the right place.
For figure 1E and F the statistics performed should be indicated.

Limitations

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instead of autism write of NHE9 variants
Another limitation is using HEK cells and not CNS cells types.

Methods

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Did the authors do transfection control with empty plasmid? How transfection efficiency was controlled?
Authors missed describing DAPI staining.
Usually for centrifugation g should be given and not rpm which is dependent on the machine used.
Western, DTT treatment is not mentioned as in figure 1.- densitometry analysis of bands as indicated above would be helpful.
From where are the antibodies for Tubulin and GAPDH? Also the used dilutions of all antibodies is necessary.
Statistical analysis description is missing, even if non-significance was found.